Project Summary Ulcerative colitis (UC) is a common, chronic form of inflammatory bowel disease characterized by superficial inflammation and damage to the epithelium of the colon and rectum. While causes of UC are unknown, genetic studies point to inappropriate immune signaling, mishandling of the gut microbiota, and altered epithelial repair mechanisms as contributing factors. A common feature of UC is an altered mucus layer in the colon epithelium, which may be due to changes in mucin biosynthesis as well as to defects in differentiation of goblet cells. As the mucus layer normally serves as a protective innate immune barrier, separating microbes from the epithelial surface, defects in mucus assembly allow gut bacteria to interface with the colonic epithelium more closely. This has been implicated in triggering immune responses or increasing susceptibility to infections that contribute to UC pathophysiology. ER stress and the unfolded protein response is intimately linked to secretory cell function and inflammation in the intestinal epithelium. The intestinal epithelium and other mucosal epithelia are unique in that they express an additional ER stress sensor called IRE1?. IRE1? appears to protect against colonic injury and inflammation by unknown mechanisms. We recently found that IRE1? functions in the unfolded protein response in intestinal epithelial cells, and discovered that the colon of IRE1?-/- mice closely resembles human UC. IRE1?-/- mice have fewer mature goblet cells in the colon compared to IRE1?+/+ littermates. This is associated with a nearly abolished inner mucus layer that places the gut microbiota immediately adjacent to the colonic epithelium. Under steady state conditions IRE1?-/- colonic crypts do not express elevated markers of a UPR, suggesting that unresolved ER stress cannot explain this phenotype. Instead, it appears that IRE1? is required for expression of transcription factors that specify goblet cell development. Notably, we find IRE1? expression and assembly of the colon mucus layer are stimulated by the gut microbiota. These data place IRE1?'s function at the interface of the microbiota and the epithelium, regulating the innate barrier that protects the host from inflammatory signals and infection. The purpose of this grant is to understand how IRE1? protects against colitis, where our central hypothesis is IRE1? protects against colitis by controlling microbiota-induced goblet cell differentiation and assembly of the colon mucus layer. We will use in vitro and in vivo models to understand how the microbiota regulates IRE1? function (Aim 1), define how IRE1? regulates goblet cell differentiation (Aim 2), and establish the relevance of IRE1? function in protecting against colitis in experimental models and human UC (Aim 3).